Telomerase is the ribonucleoprotein complex responsible for maintaining telomeres, the long stretches of tandem repeating DNA and associated binding proteins at the ends of linear chromosomes. Telomeres protect the ends of chromosomes from being recognized by the cellular DNA repair machinery as double strand breaks and also prevent end-to-end joining. In the absence of active telomerase, telomeres shorten with each round of DNA replication. Once a critical amount of the telomere is lost, the cell enters replicative senescence and sometimes undergoes apoptosis. Telomerase expression in most somatic cells is undetectable, with the opposite true of most cancer cells-allowing them to replicate indefinitely. This difference in activity makes telomerase an attractive target for ani-proliferative drugs. The goal of this fellowship will be to acquire a cryoelectron microscopy structure of the Tetrahymena thermophila telomerase holoenzyme complex, assign each component, deduce function from structures obtained at different points in the catalytic cycle, and solve the structure of the p19 subunit by NMR and X-ray crystallography. During replication, the chromosomes of T. thermophila fragment into ~20,000 subchromosomes, each with their own telomeres. To maintain so many telomeres, T. thermophila also expresses a large amount of telomerase. This has ensured its use as a model organism. The specific aims are to (1) purify T. thermophila telomerase holoenzyme from TAP-tagged strains for electron microscopy and obtain a negative stain structure, (2) localize individual protein and RNA subunits by antibody and affinity labeling, (3) optimize sample conditions for cryoelectron microscopy and determine a cryoelectron microscopy structure, (4) determine the structure of Tetrahymena telomerase holoenzyme with bound DNA primer and compare to the unbound structure, and (5) Use NMR and crystallography to determine the structure of the T. thermophila protein subunit p19. These experiments will reveal the complete structure of a telomerase holoenzyme as well as offer insight into what roles intermolecular contacts and subunit movement play in telomere catalysis.